Dengue is considered as one of the main public health problems worldwide, among the diseases caused by arbovirus, due to its importance in terms of morbidity and mortality in the human population, particularly in tropical and sub-tropical regions. Its etiological agent is the dengue virus which is transmitted by the Aedes genus, generally Aedes aegypti. 
The dengue virus (DENY) belongs to the Flavivirus genus, of the Flaviviridae family. There are 4 antigenically and serologically distinct types of virus: DENV1, DENV2, DENV3 and DENV4. Although the primary infection by the dengue virus induces to immunity to the infecting serotype, there is no long-lasting cross protection against the other virus serotypes, leading to sequential infections with the different dengue viruses.
The dengue virus is composed by a viral envelope and a nucleocapsid complexed to a RNA molecule. Its genome comprises approximately 10,700 nucleotides and is constituted by a single-stranded RNA with positive polarity which codifies the precursor polyprotein of flaviviral proteins. This precursor is clived by cellular proteases and by viral protease generating three structural proteins, C (capsid), prM (pre-membrane) and E (envelope), and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The translated structural proteins are incorporated in mature infectious virions, while non-structural proteins are involved in virus replication and development.
Dengue virus E protein (Envelope) is a structural and transmembrane protein, being glycosylated in almost all the Flaviviruses. This E protein is the main glycoprotein component found in the surface of these viruses, having a molecular weight of 55 to 60 kDa, constituted by 493 to 501 amino acids.
The E protein is associated to a number of important biological activities. The E protein acts as a binding protein, interacting with the receptor found in the cellular surface and mediating the fusion of the virus membrane with the host cell membrane. This protein is also related to the nucleocapsid dissociation and plays an important role in virus virulence. In addition, the E protein is a strong immunogen, generating high antibody levels in patients infected with the virus, which can be neutralizing ones, binding to epitopes which interact with cellular receptor and preventing, thus, the virus from entering in the host cells (Chang, 1997; Kinney & Hung, 2001).
The E protein is formed by an elongated dimer which extends parallel to the virus membrane. Each monomer is composed by three domains: I, II and III. Domain I is a central one, comprising 120 amino acids residues: 1-51, 133-193, 279-296. Domain II is elongated, comprising the region where the two monomers are linked in several points in the molecule, forming the dimer. Domain II comprises the following amino acids residues: 52-132, 194-280. Domain III is located in the carboxy-terminal portion, it presents as an immunoglobulin, whose function is to bind to cell receptors, such as, for example, DC-SIGN present in immature dendritic cells. The virus/cell binding mediated by domain III promotes the viral particle endocytosis. Domain III comprises the amino acids residues: 298-394. The epitopes present in this region are able to induce type- and subtype-specific neutralizing antibody response.
A number of vaccines have been proposed for the fight against dengue, however, none of the suggested approaches presented the required characteristics for a mass vaccination, such as safety and a long-term protective immune response against the different circulating viral serotypes.
One of the main difficulties is to develop a vaccine prototype containing components inducing a protective immunity against the four dengue virus serotypes, without generating consequences such as hemorrhagic fever, and using the lowest number or doses as possible.
Considerable efforts have been performed for the development of an attenuated 4-valent vaccine. The most promising of these candidates consists in viruses attenuated through passages in cell cultures. Such vaccine is in the clinical evaluations phase, however, some complications have already been noted. Moreover, there is potential risk of serious infections by viruses which can raise from gene reversions or recombination, being difficult to formulate a multivalent live attenuated vaccine due to the possibility of homologous or heterologous interference during the viral replication (Barrett, 2001; Kinney & Huang, 2001).
Another strategy used for the development of a vaccine against the dengue virus is the construction of chimeras. This technology is being used in a number of researches of vaccines against Flavivirus, including the Dengue virus (Galler et al., 2005).
Despite of the advances obtained so far, there is still a great challenge: to produce a vaccine which is able to generate a homogeneous response against the four Dengue virus serotypes.
The DNA vaccine represents an efficient technology in the development of vaccines for the control of infectious agents. This technique comprises the inoculation of an eukaryotic expression plasmid containing the antigen of interest, which is synthesized in vivo by the inoculated organism cells and presented by the histocompatibility complexes I and II (MHC I and II), activating a specific immunity. The endogenous expression of the antigen by the host cells seems to simulate a natural viral infection being able to generate both a humoral immune response, with antibody production, and a cell response, with induction of cytotoxic T lymphocytes. The induction of these two forms of immune response provides advantages compared with the subunit vaccines, which mainly or exclusively produce an antibody response and is compared to the cellular response of live attenuated vaccines, without presenting the risk of reversion to the pathogenic form of the infectious agent. In addition, the DNA vaccines are stable to temperature ranges, of lower cost for large-scale production and allow a rapid selection of sequences to be evaluated.
Recently, several groups have analyzed the use of DNA vaccine in the flavivirus control. Some of these studies with different viruses from this family have shown the induction of a flavivirus-specific protective immune response in mice and non-human primates following vaccination with plasmids codifying flaviviral proteins prM and E (Phillpotts et al., 1996; Kochel et al., 1997; Raviprakash et al, 2000). Some analysis were also conducted using DNA vaccines containing flavivirus non-structural protein genes, such as: the Japanese encephalitis, hepatitis C and dengue viruses (Lin et al., 1998; Encke et al., 1998; Wu et al., 2003; Costa et al., 2006, 2007).
Recent studies showed that DNA vaccines containing the full sequence of the genes codifying proteins prM and E, and DENV1-4 virus domain III region, were able to induce a humoral immune response with the presence of antibodies against Dengue in murine models. However, in these studies the protective potential of the DNA vaccines was only indirectly evaluated (seroneutralization and cell response), no direct challenge tests were conducted with lethal doses of the Dengue virus to evaluate the efficacy of such vaccines (Chen et al., 2007). Some DNA vaccines against Dengue were tested in non-human primates (Aotus and Rhesus) and provided partial protection, evaluated through viremia in animals challenged with the Dengue virus (Raviprakash et al., 2000; Putnak et al., 2003; Blair et al., 2006).
Concerning the immunization method, the present inventors emphasize that Putnak et al (2003), developed a DNA vaccine containing the genes which codify proteins prM and E from DENV2, observed the immunogenicity of it in mice and subsequently immunized Rhesus monkeys. However, several vaccine doses were required to induce a protective and short-lasting response. Seven months following the immunizations, the animals were shown to be no longer protected against the challenges with the dengue virus. Thus, we can note that these studies indicate the need to use new strategies to increase the length of the immune response. More recently, it has been shown that the combination of DNA vaccines with other vaccines can generate more efficient immune responses than the same approaches isolately.
Patent application WO2008/127307, published on Oct. 23, 2008, is directed to an immune response induction method against the dengue virus using a prime/boost vaccination method. More specifically, document WO2008/127307 is directed to the administration of a dengue virus immunogen dose comprising a DNA vaccine or a 4-valent DNA vaccine or a 4-valent purified inactivated vaccine and a booster dose of a dengue virus immunogen, comprising a 4-valent attenuated virus vaccine. However, such strategy presents the difficulties inherent to the use of a 4-valent attenuated dengue virus vaccine, such as for example the possibility of interference of one or more viruses in the in vivo replication of the other viruses, thus leading to heterogeneous immune responses in relation to the different virus serotypes. In addition, the document is also directed to the use of adenovirus containing genes codifying dengue proteins as a reinforcement for a 4-valent vaccine, what may imply in low efficiency in inducing immune responses against dengue in humans. This fact is due to the presence, in humans, of previous immune responses against adenovirus, which may prevent the infection with vector adenovirus carrying dengue virus genes and, consequently affecting the efficiency of the immune response to dengue virus.
It is also known the patent application US20080193477, published on Aug. 14, 2008, a method employing an immunization regime comprising the administration of a first yellow fever vaccine followed by the administration of a chimeric flavivirus-based dengue vaccine. However, other reports in the literature showed that the immunization with the yellow fever vaccine virus can affect the efficiency of the immune response generated subsequently with yellow fever chimeric viruses containing dengue virus genes. In this case, the neutralizing antibodies levels against dengue virus were significantly lower in monkeys previously vaccinated against yellow fever (Galler et al., 2005). As discussed herein, we verify that despite of the efforts which have been developed, there is still a need for the development of more efficient DNA vaccines and able to induce an increased immune response, as well as the use of combined vaccine strategies enhancing such immune response.